The present disclosure relates generally to processes for the stabilization of organosulfur equivalents derived from Allium tissues in a form that retains biological activities relevant to human health promotion. More particularly, Allium tissues are specifically combined and reacted to yield thiosulfinate preparations for further utilization in the preparation of mixed disulfide conjugates with thiol-containing residues.
Epidemiological studies having consistently shown that the consumption of Allium vegetables (such as onions, garlic, chives, leeks, and the like) have been associated with reduced incidence of many diseases among human populations (Kendler, 1987; Steinmetz and Potter, 1996; Ernst, 1997; Bianchini and Vainio, 2001; Tapiero et al, 2004). The health benefits of Allium vegetables are widely attributed to or correlated with the enzyme-transformed organosulfur compounds called thiosulfinates (i.e., fresh-like Allium flavors), sulfides (cooked-type Allium flavors) and related species.
Specifically, it has been found that thiosulfinates have biological activities related to health-benefits of cancer chemoprotection, anti-inflammatory, antioxidant, and antimicrobial effects, and cardiovascular benefits of cholesterol-, lipid-, and platelet activity-lowering effects, among others (Lawson, 1998; Tapiero et al., 2004). For example, various studies have shown that the administration of fresh garlic, etheric extracts, or its active component allicin (thio-2-propene-1-sulfinic acid) resulted in beneficial effects on cardiovascular risk factors, mainly hyperlipidemia and thrombogenesis in humans. Furthermore, garlic was shown to increase fibrinolytic activity, inhibit platelet aggregation, and reduce serum cholesterol levels. Additionally, garlic juice was shown to inhibit the growth of bacteria of the genera Staphylococcus, Streptococcus, Vibrio and Bacillus, and of zoopathogenic fungi and many strains of yeast, including Candida albicans. 
Thiosulfinates in the diet are virtually 100% absorbed by the human body (Lawson and Wang, 2005). The details of their metabolic fate, however, are not known other than that a stoichiometric amount of allyl methyl sulfide is formed from allicin, which provides the basis for determining bioavailability. Furthermore, thiosulfinates in these vegetables have limited stability in fresh minced tissues (Block et al., 1992), and chemically evolve into other organosulfur compounds, principally (poly)sulfides, γ- and α-sulfinyl disulfides, vinyl dithiins, as well as other lesser abundant derivatives, some of which also have limited stability. These subsequent chemical transformations may be evoked by elevated temperature, pH adjustment and the addition of organic solvents to tissue macerates and/or extracts (Block, 1992). In the particular case of bulb onion (A. cepa) and related species rich in S-1-propenyl-L-cysteine sulfoxides (1-PeCSO), much of the alliinase-transformation products of 1-PeCSO is lost as propanethial-5-oxide (PTSO) from tissue due to volatility (see FIG. 1); thus this type of instability also leads to a lack of retention of organosulfur equivalents within the tissue matrix and hence, a likely depletion of any health benefits associated with onion consumption. Since PTSO is also the pungent component in freshly cut onions that induces tearing, the presence of PTSO (also called the lachrymatory factor or “LF”) is a major reason why some people find fresh-cut raw onions objectionable.
Because of the limited stability of many Allium organosulfur components, and uncertainly about which are the most health-relevant agents in intact or processed Allium vegetable tissues, efforts have been made to manage the biochemistry of organosulfur evolution in Allium vegetable products, both for use as foods and dietary supplements. One approach has been to prepare an immobilized alliinase to react with synthesized S-allyl-L-cysteine sulfoxide (2-PeCSO) to generate the major garlic thiosulfinate, allicin (Mirelman et al., 1997; Miron et al., 2006). The disadvantages of this approach, however, include use of synthetic substrate (2-PeCSO), and the expense of moderately sophisticated technology, concern about long-term stability of the immobilized enzyme, and projected scaling-up of allicin synthesis.
Another approach has made use of aged garlic preparations, where sliced cloves are extracted by prolonged incubation in aqueous ˜20% ethanol (Lawson, 1998). The principal organosulfur components that evolve are the S-alk(en)yl-L-cysteine (CySR) and S-alk(en)yl-L-mercaptocysteine (CySSR) species, at a mass ratio of about 5:1, where R is predominantly the allyl group. The resulting product is an aged garlic extract (Kyolic®), marketed as a dietary supplement. Although this product is prepared by rather simple steps, the reported time required for aging is greater than 10 months (Lawson, 1998) (20 months stated on Wakunaga/Kyolic® website accessed 2 Sep. 2009), and the CySR and CySSR organosulfur chemotypes have not been studied for potential health benefits as much as the thiosulfinates and (poly)sulfides; thus, the impact that aged garlic extract may have on disease risk reduction and which components are responsible is not well established by comparison.
Additional approaches including conventional breeding and other molecular biology strategies have also been used to manage the evolution, fate and profile of organosulfur components in Allium tissues.
Accordingly, there is a need in the art for developing the means to manage and control the transformation of thiosulfinates and related organosulfur species into stabilized, naturally occurring derivatives retaining biological activity relevant to human health promotion. Additionally, it would be advantageous if the organosulfur equivalents (also referred to herein as mixed disulfide conjugates) could be prepared in a short period of time using simple means.